Polarization Hybridization Assays

polarization measurements do not require sqmration steps. 

NtR dyes excitable at 770 nm, one can expect to see the concept of fluwogeiuc PCR substrates extended to longer excitation and emissiMi wavelen hs. PCR can also be quantified by dyes which bind to double stranded DNA and by RET.  

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Nakagami et al. (1991) proposed a different universal probe system. In their procedure, phagemids are used to obtain strand-specific ss DNA molecules, one with the insert complementary to the target sequence and a second without the insert and with the opposite polarity (Fig. 7.23). The ss DNA without insert is labeled with any label (usually a secondary label) and this labeled DNA is hybridized with the complementary strand containing the insert. This leaves only the probe sequence ss whereas the tag sequence is ds. This method has several advantages (i) like the sandwich method it leaves the probe sequence single-stranded (ii) hybridization of the tag sequences in solution instead of on the solid phase as in a sandwich assay is much faster (iii) no fragmentation is required. 

DASH, dynamic allele-specific hybridization AS-PCR, allele-specific polymerase chain reaction AS-PE, allele-specific primer extension APEX, arrayed primer extension FP-TDI, fluorescence polarization template directed dye terminator incorporation MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry OLA, oligonucleotide ligation assay RCA, ... 

Current methods combine at least one of four different principles of allelic discrimination (hybridization, primer extension, ligation, or restriction) with one of four different detection techniques (chemiluminescence/ fluorescence, fluorescence polarization, resonance energy transfer, and mass spectrometry). Assay formats range from (slab)- gel electrophoresis, plates, particles, fibre arrays, and microchip arrays to semi- and homogenous assays that do not require any further sample separation or purification. 

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